Wound dressings, methods and apparatus for making same and storage and use thereof

ABSTRACT

Provided is a wound dressing prepared from whole blood that is clotted ex vivo to form a sheet of clotted blood that is applied onto the skin over a sight of skin injury.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/145,205, with a 371 (c) filing date of Sep. 14, 2011, which is aNational Phase Application filed under 35 U.S.C. §371 as a nationalstage of PCT/IL2010/000066, filed on Jan. 27, 2010, an applicationclaiming the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 61/147,513, filed on Jan. 27, 2009, the content of eachof which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to wound dressings, as well as to methods andapparatus for producing and/or using the dressings, storage of thedressings and use of the dressings.

BACKGROUND OF THE INVENTION

The skin is the largest organ of the mammalian body and serves severalcritical functions. In some cases, the skin is disrupted by trauma (e.g.laceration, abrasion, burns or puncture) or by ulceration (e.g. diabeticfoot ulcers). Disruptions in the skin which do not heal, orspontaneously recur, are known as chronic wounds (Fowler (1990) inChronic wounds: an overview. In: Krasner D, editor. Chronic wound care:a clinical source book for healthcare professionals. King of Prussia,Pa.: Health Management Publications, Inc;. pp. 12-8 and Singh et al.(2004) Asian J Surg 27:326-32).

Approximately 1% to 2% of individuals will be affected by leg ulcerationduring their lifetime, and this figure will likely increase as thepopulation ages (Rees and Hirshberg (1999). Adv Wound Care 1999; 12:4-7and Callam M. (1992) Phlebology 7:S6-S12). Global wound care expensesreaches up to $13 to $15 billion annually (Walmsley (2002) In: Clinicalreports. London: PJB Publications, Ltd.).

Patient groups suffering chronic wounds include, but are no limited to,diabetic patients, geriatric patients and patients with circulatoryproblems. Also, chronic wounds can appear as a result of acute trauma oras a post surgery symptom.

Chronic wounds may vary in size, depth and stage of healing. Wounds cancontain necrotic tissue, infection, scabs, or exudates (purulent,cerotic).

Chronic wounds can be classified by their cause such as pressure,diabetic, ischemic, venous, and tear and/or by the nature of the wounditself such as its depth and/or stage of healing and/or discharge and/orinfections. Burns are another wound type which is difficult to treat.

Conventional burn treatment typically relies upon a topical antibioticcream (e.g. Silver sulfadiazine) followed by a non-stick dressing andgauze. Use of biologic dressings based upon cultured cell grafts and/orfractionated blood products has also been suggested. According todifferent burn management strategies, frequency of dressing changes canvary from twice per day to about once per week.

A number of non conventional burn treatment strategies have beenproposed. A non-exhaustive overview of some of the art follows.

WO/2001/021195 describes a dressing including preformed fibrin thatfunctions as a non-adhesive covering of a burned skin surface as well asfunctioning as a delivery vehicle for pharmaceutical compounds that areentrapped within the fibrin clot.

J. Travis (Science News Online 155 (25); Jun. 19, 1999) describes fibrinbandages produced by Martin MacPhee at the American Red Cross' HollandLaboratory in Rockville, Md. The bandages employ a cloth made ofbiodegradable material saturated with thrombin, fibrinogen, and factor13 purified from human blood. The bandages are brittle until they getwet, and then they become flexible.

US 2002/0146446 describes a surgical-medical dressing which uses asandwich of two extracellular matrices grown on a composite composed ofgelatin-fibronectin-heparan sulfate. The culture medium used to grow thetwo cell types (dermal fibroblasts and dermal microvascular endothelialcells forming the second extracellular matrix) is the conditioned medium(CM) obtained from human umbilical endothelial cells used to form thefirst extracellular matrix. All cells in tissue culture are detachedleaving their secreted acellular matrix behind and intact. This CM canalso neutralize the enzyme DISPASE commercially used to detach culturedepithelial sheets (“Cultured epithelial autografts” (CEAs)) from thematrix on which the human epidermal cells, forming the sheets are grown.CEAs are clinically used in wound and burn management

Henderson L. et al, discloses the healing of superficial skin burns by aautologous platelet gel dressing. (Ear, Nose & Throat Journal, 2003).

U.S. Pat. No. 3,723,244 describers a method of producing fibrin in sheetform by centrifuging an aqueous dispersion of fibrin wherein monomericfibrin molecules are combined by polymerization to form strands offibrin. The centrifuging step is conducted in a vessel having a wall forinterception of particles undergoing centrifugal acceleration thereinand at a speed pelletizing on said wall the strands of fibrin resultingfrom polymerization, so that the pelletized strands interlock to form asheet which is recovered from the wail. The aqueous dispersion can beblood plasma and the resultant fibrin sheets are described as useful asa dressing for burns.

U.S. Pat. No. 6,521,265 describes a method of promoting healing of ableeding wound including mixing a substantially anhydrous compound of asalt ferrate, which hydrates in the presence of water to produce Fe⁺⁺⁺and oxygen, combined with an insoluble cation exchange material, with aquantity of an aqueous media such as whole blood taken directly from thewound with deionized water; aqueous sodium chloride; aqueous dissolvedgelatin; aqueous carboxy methacel; and aqueous carbohydrate solution toform a spreadable paste. The paste is applied to the wound within ashort working time to promote blood clotting. According to thedescription, the presence of oxygen, substantially reduces the level ofbacteria, virus and fungus at the wound as a protective coating formsover the wound.

U.S. Pat. No. 4,347,841 describes biological dressing for burn woundsformed by removing free hemoglobin from a red blood cell concentratewhich is subjected to hemolysis. The dressing contains the stroma,subcellular elements and precipitated protein from the human red bloodcorpuscle concentrate freed from the hemoglobin and can be used in apulverulant or layer form with, if desired, an appropriate support.

US 2007/0275461 and US 2004/0171145 describe artificial dermis obtainedfrom plasma with platelets and human fibroblasts. The plasma withplatelets is obtained from fractionating whole blood from the patient bylight centrifugation, and the human fibroblasts are obtained from a skinbiopsy. Clotting is obtained by adding calcium. The artificial dermis isdescribed as providing for rapid growth of keratinocytes seeded on itssurface to build an artificial skin which can easily be transplanted.Large areas of artificial dermis are described as being obtained from asmall skin biopsy and minimal quantities of plasma with platelets. Theartificial skin is described as useful to treat major burns, chronicskin ulcers, or be used with genetically altered cells as a vehicle forgene therapy.

US 2004/0124564 describes a process for the preparation of a chemicallymodified fibrin-fibrillar protein (FFP) composite sheet for medicalapplication and the FFP composite prepared thereby. According to thedescription, the FFP sheet finds potential use as a dressing aid in thetreatment of various external wounds including burn wounds.

Use of purified or fractionated blood components in other medicalcontexts is also described. A non-exhaustive overview of some of the artfollows.

Khalafi et al. (Eur J Cardiothorac Surg 2008; 34:360-364.) describesapplication of platelet rich and platelet poor plasma to significantlyreduce occurrences of chest wound infection, chest drainage, and legwound drainage in coronary artery bypass grafts.

Medtronic, Inc., Minneapolis, MV; USA produces devices for processingautologous blood to concentrate platelet-rich plasma into an autologousplatelet gel for use in surgery for improving tissue healing.

Some commercially available wound care products use blood fractionsand/or factors isolated from blood.

For example, a variety of growth factors have been found to play a rolein the wound healing process, including platelet derived growth factor(PDGF), epidermal growth factor, fibroblast growth factors, transforminggrowth factors, and insulin-like growth factor. Various wound healingtechnologies based on fractionated blood or blood derived growth factorsare commercially available.

One commercially available technology employs recombinant DNA techniquesto create purified growth factors. REGANEX™ Gel (manufactured bySystagenix Wound Management) is a topical gel, containing the activeingredient becaplermin with an activity similar to that of humanplatelet derived growth factor (PDGF). This recombinant growth factor isproduced by recombinant DNA technology by insertion of the gene for theB chain of platelet-derived growth factor (PDGF) into Saccharomycescerevisiae.

Another commercially available technology employs platelet rich plasma(PRP). PRP is isolated from whole blood by centrifugation. AutologousPRP contains a mixture of activated growth factors cytokines andchemokines, with reduced potential for immune response. Exposure of PRPto a solution of thrombin and calcium chloride results in thepolymerization of fibrin from fibrinogen, creating a platelet gel whichcan then be applied to wounds. The PRP provides the wound with growthfactors, chemokines and cytokines that promote angiogenesis and regulatecell growth and formation of new tissue. AutoIoGel™ (manufactured byCytomedix, Inc.) and SafeBlood® (manufactured by SafeBlood Technologies)are two autologous blood-derived products that can be prepared at thebedside for immediate application. Both AutoioGel™ and SafeBlood® havebeen specifically marketed for wound healing.

Another commercially available technology employs fibrin glues orsealants with hemostasis and gluing properties which enhance woundhealing. Commercial fibrin glues are created from pooled homologoushuman donors. TISSEEL™ (manufactured by Baxter) is an example ofcommercially available fibrin sealant. The action of this productallegedly simulates key features of the physiological process of woundclosure. The product contains a highly concentrated fibrinogen aprotininsolution, which among other ingredients contains Factor XIII, and asolution of thrombin and calcium chloride are applied to the wound area,where the mixture coagulates. The presence of Factor XIII causes thefibrin to crosslink, which gives the coagulum additional resilience.

SUMMARY OF THE INVENTION

A broad aspect of the invention relates to treatment of skin injuriesusing clotted blood. The clotted blood is formed so it can be appliedonto a skin injury.

The term “whole blood” should be understood as referring to (i) bloodtaken from venous or arterial circulation; (ii) (ii) blood that has notbeen modified except for the addition of an anticoagulant or otherchemical or biological substances or removal of one or more bloodcomponents (e.g. a portion of the plasma), where such modification doesnot affect its overall physical or chemical characteristics; or (iii)blood containing all its components, such as red and white blood cells,platelets and more, particularly all components required for clotformation. It should be noted that the term “whole blood” does notnecessarily mean that the blood used as a source for the clotted bloodis identical to the blood withdrawn from an individual. At times, thewithdrawn blood may be subject to procedures such as dialysis, passagethrough a column, etc., in order to remove certain, e.g. undesired,component before being used in therapy of skin injuries according to theinvention (an example is the removal of certain immunogenic or anti-seracomponents in the case of the use of a non-autologous blood). Also,according to some exemplary embodiments of the invention the whole bloodis supplemented with one or more additives. Additives include, but arenot limited to coagulation influencing additives, physical propertychanging additives and medically active additives.

Skin injuries treatable in accordance with the invention include allkinds of injuries of the skin where skin is missing or where the therapytargets, among others, skin re-growth. These include both non-bleedingwounds (e.g. burns and ulcers) and bleeding wounds (e.g. abrasions).

Coagulation influencing additives include, but are not limited toanticoagulants, anti-anticoagulants and/or coagulation accelerators(such as negatively-charged phospholipids (PL) and surface contactagents as Kaoline). In order to avoid uncontrolled blood clotting, oneor more anticoagulants (e.g. EDTA) may be added to the blood immediatelyupon withdrawal from an individual. Anticoagulants are in fact typicallyincluded in test tubes or other containers where the blood is collected.Also, blood stored in a blood bank typically includes one or moreanticoagulants. According to some exemplary embodiments of the inventionin order to counteract the anticoagulant in the whole blood preparation,an anti-anticoagulant is added to permit the blood to clot. For example,Ca²⁺ ions can be used to counteract the activity of EDTA. These ions canbe added as a solution of a soluble calcium salt. Since the type andamount of anticoagulant added to a unit of whole blood are typicallyknown, it is possible to select an appropriate anti-anticoagulant andcalculate an appropriate amount to use.

Physical property changing additives include, but are not limited to,diluents (e.g. saline solutions), hardeners (e.g. minerals),plasticizers and elasticizers.

Medically active additives include but are not limited to antibiotics,antiseptics, analgesics, growth factors, cytokines, enzymes, hormones,cells, anti-inflammatory agents and many others as may be envisaged byone of ordinary skill in the art of wound care.

Use of medically active and physical property changing additives isdescribed in US 2004/0147024, U.S. Pat. No. 5,610,148 and U.S. Pat. No.5,629,287 the relevant content of which being incorporated herein byreference.

Some exemplary embodiments of dressing as disclosed herein are basedupon the realization that a blood clot naturally contains the physical,chemical and functional properties which make it an effective dressingto cover the wound and promote healing. Without wishing to be bound bytheory, it is believed that clotted whole blood must necessarily addressall basic requirements of a wound dressing. Optionally, by using anautologous blood clot (a clot from whole blood extracted from thepatient) any potential for an immune response is greatly reduced.Optionally, when using a homologous blood clot (namely a clot from wholeblood extracted from a different individual, e.g. obtained from a bloodbank), the potential for immune response can be reduced by serologicaltyping.

An aspect of some embodiments of the invention relates to formation of alayer or sheet of clotted whole blood on a support structure. Accordingto some exemplary embodiments of the invention the support structure isembedded within the clotted whole blood. Alternatively or additionally,the support structure is external to the clot. According to someexemplary embodiments of the invention the support structure aids intransfer of the clotted whole blood from one location to another and/orcontributes to a change in contraction behavior during clotting and/orcontributes to a change in physical properties of the resultant wholeblood clot (e.g. increased strength). An example of a support structureis a pad of a fibrous material such as gauze.

An aspect of some embodiments of the invention relates to concurrentformation of a plurality of sterile whole blood clots from a source ofwhole blood. According to some exemplary embodiments of the inventionthe clots are formed in sheets of a desired thickness. Optionally, thesheets in the plurality have a common thickness. Optionally, the sheetsare formed on a support structure.

An aspect of some embodiments of the invention relates to a sterilesheet of clotted whole blood including an anticoagulant and sufficientanti-anticoagulant to permit clotting. According to some exemplaryembodiments of the invention the sterile sheet of clotted blood isprovided in a package or wrapper which maintains sterility duringstorage and/or transit. Also the blood clot is typically formed as asheet, the invention is not limited to blood clots formed into such aspecific form.

An aspect of some embodiments of the invention relates to application ofclotted whole blood to a skin surface of a subject. According to variousexemplary embodiments of the invention the clotted whole blood can beapplied as part of a wet dressing or a dry dressing. According to someexemplary embodiments of the invention the skin surface includes a wound(e.g. ulcer or laceration) and/or a burned skin surface.

Optionally, the clotted whole blood is applied with an internal and/oran external support as described hereinabove and hereinbelow. Accordingto some exemplary embodiments of the invention the clotted whole bloodis covered with a conventional dressing. The term “conventionaldressing” as used in this specification and the accompanying claimsrefers to a covering including one or more layers each independentlyselected from a non adhesive layer, an absorbent layer (e.g. gauzeand/or non woven fabric or similar mesh) and an adhesive layer (e.g.tape).

An aspect of some embodiments of the invention relates to a bank ofdressings prepared from units of clotted whole blood. According to someexemplary embodiments of the invention the dressings are catalogued byunique identifiers corresponding to the units. Optionally, the uniqueidentifiers are used to match a subject to one or more specificbiological dressings.

Optionally, matching information is provided as machine readable dataassociated with said unique identifier in a look up table in a memory ofa computer. Machine readable data includes, but is not limited to, datarelated to blood type, cross-match data, HLA haplotype data and othergenetic data. Alternatively or additionally, matching information isprovided as an aliquot of un-clotted blood reserved from each of saidunits of whole blood from which each of said plates in said pluralitywas prepared, each aliquot associated cataloged by said uniqueidentifier. “Blood type” as used herein refers to any antigen and/orantibody characterization and is not limited to A; B; AB; O and Rh^(+/−)

An aspect of some embodiments of the invention relates to spreading anamount, e.g. a unit of whole blood obtained from a donor or blood bankover an area, e.g. 50-10,000 cm², and allowing the blood to form a clot.According to some exemplary embodiments of the invention sterility ofthe blood is maintained during clotting. As can readily be understood,the area measures noted above are exemplary and they may be smaller orlarger. There may, at times, be a tradeoff between strength of theresultant blood clot sheet and area, with larger areas producing weakerclots. According to some exemplary embodiments of the invention asupport layer contributes strength that permits spreading of the bloodover a larger area. In general, the thickness of blood is reduced duringclot formation by weeping or sweating of plasma from the clot duringand/or after formation. For example, a 3 mm layer of whole bloodproduces a clot with a thickness of about 1.2 mm after 1 day in vitro.According to some exemplary embodiments of the invention a coagulationinitiator is added prior to spreading. Optionally, the coagulationinitiator is selected to neutralize activity of an anticoagulant in theblood.

An aspect of some embodiments of the invention relates to use ofunfractionated whole blood in manufacture of a wound blood dressing.

An aspect of some embodiments of the invention relates to application ofwhole blood to an area of skin in need of treatment to form a blood clotthereon. According to some exemplary embodiments of the invention thearea is covered by an absorbent layer. Optionally, the absorbent layerincludes a mesh such as gauze and/or non-woven fabric. Optionally, theclot is covered after formation. According to some exemplary embodimentsof the invention burned skin is treated. Optionally, sterility of theblood is maintained during application.

An aspect of some embodiments of the invention relates to cutting slicesor sheets of clotted whole blood from a block of clotted whole blood.The term “block” as used in this specification and the accompanyingclaims should be interpreted broadly so that it includes all threedimensional shapes.

As used in this specification and the accompanying claims, the adjective“removable” indicates the described object has been engineered to allowremoval by an average user without undue effort. Removable includes bothitems which are removed without tools, and those that require use of atool (e.g. scissors, knife or opener). In some exemplary embodiments ofthe invention, tools for removal are provided with their matchingremovable parts in the manner of a sardine can with an attached “key”.

According to an embodiment of the invention there is provided a devicefor clotting whole blood and forming it into a clot for use in treatmentof a skin injury. The device has a blood receptacle for receive theblood, the receptacle being shaped in a manner so as to impart a desiredfinal shape, e.g. planar, on the formed blood clot.

According to some exemplary embodiments of the invention a device forclotting whole blood ex-vivo is provided, the device comprising: (a) aninlet including a channel of fluid communication to an interior of thedevice; (b) a sample receptacle adapted to spread a sample providedtherein to produce a uniform layer; and (c) at least one supportstructure deployed in the receptacle so that the uniform layer forms onthe structure. It optionally includes, also (d) a removable covercovering the receptacle and adapted to maintain sterility of the layer.

Optionally, the support structure includes an external layer.

Optionally, the support structure is adapted to become embedded in thelayer.

Optionally, the layer is constructed of a material which adheres to ablood clot formed thereupon.

Optionally, the support structure includes at least one materialselected from the group consisting of nylon, polyurethane, cotton,cellulose, a silicon and rubber.

Optionally, the receptacle is constructed of at least one materialselected from the group consisting of glass, cellulose, polystyrene,polyurethane, polyvinylchloride, polycarbonate and a rubber.

Optionally, the receptacle contains a sufficient amount of ananti-anti-coagulant to cause coagulation of blood including the sample.

Optionally, the interior of the device is under vacuum pressure.

According to some exemplary embodiments of the invention there isprovided a device for clotting whole blood ex-vivo, the devicecomprising: (a) at least one inlet including a channel of fluidcommunication to an interior of the device; (b) a plurality of samplereceptacles interconnected to allow concurrent inflow from a commonsource, each of the receptacles adapted to spread a sample providedtherein to produce a uniform layer, e.g a sheet; (c) a sterility guardadapted to maintain sterility of the layer in each of the receptacles.

Optionally, the device includes a support structure deployed in each ofthe receptacles so that the layer forms on the support structure.

Optionally, the support structure includes an external layer constructedof a material which adheres to a blood clot formed thereupon.

Optionally, the support structure includes a mesh embedded in the layer.

Optionally, the interconnection is in at least one configurationselected from the group consisting of in parallel and in series.

Optionally, the receptacles are arranged in a stack.

Optionally, removal of one of the receptacles does not affect sterilityof the layer in any other receptacle.

Optionally, the sterility guard includes a cover associated with atleast some of the receptacles.

Optionally, at least some of the receptacles serve as the sterilityguard for another of the receptacles.

Optionally, the device further includes an outlet valve providing fluidcommunication between the interior of the device and an ambientenvironment.

Optionally, the interior of the device is provided under vacuum.

Optionally, each of the receptacles contains a sufficient amount of ananti-anti-coagulant to cause coagulation of blood including the sample.

Provided by the invention is also a kit or assembly comprising one ormore of the components needed in order to prepare a blood clot-baseddressing in accordance with the invention. Such components comprise oneor more of the following: one or more blood withdrawal devices; one ormore blood collection receptacles; one or more plates, trays or othersuitable receptacles for receiving the blood and forming a clot thereon;one or more support matrices for the formed blood clot; an anticoagulantoptionally already included in the in the blood withdrawal or bloodcollection devices; an anticoagulant inhibitor (also referred to in thisspecification as anti-anticoagulant) for inducing clot formation. Thekit or assembly optionally comprises also instructions for use inaccordance with the invention.

According to some exemplary embodiments of the invention there isprovided a kit or assembly, comprising: (a) a sterile tray or plateintended for receiving whole blood; (b) an anticoagulant materialdispersed throughout the plate or included in a container and intendedfor adding to the tray or plate before or after blood is introducedtherein; and (c) an anticoagulant inhibitor to permit formation of theclot despite presence of the anticoagulant. Optionally, theanticoagulant includes at least one substance selected from the groupconsisting of EDTA (ethylenediaminetetraacetic acid), EGTA (ethyleneglycol tetraacetic acid), a citrate, Heparin and oxalate.

Optionally, the anticoagulant inhibitor is selected from Ca²⁺, Mg²⁺,negatively charged phospholipid (PL) and protamine sulfate.

Optionally, the kit or assembly is provided at ambient temperature.

Optionally, the kit or assembly is provided under refrigeration.

According to some exemplary embodiments of the invention there isprovided a method of treatment, comprising: (a) preparing a clottedwhole blood sheet; and (b) applying the sheet to an area of skin of asubject in need thereof.

Optionally, the method includes adhering a support matrix onto theclotted blood sheet.

Optionally, the method includes embedding a mesh within the clottedblood sheet.

Optionally, the matrix has a functionally significant permeability to ateast one item selected from the group consisting of a gas and a liquid.

Optionally, the matrix is constructed of at least one material selectedfrom the group consisting of nylon, polyurethane, silicon and rubber.

Optionally, the sheet is at least 1, or at least 2 or at least 3 mmthick.

Optionally, the method includes covering the sheet with a conventionaldressing.

Optionally, the method includes maintaining sterility of the clottedblood sheet from its formation until use.

According to some exemplary embodiments of the invention there isprovided a bank of biological dressings, the bank, comprising: (a) aplurality of sterile sheets of clotted whole blood, each of the platescataloged by a unique identifier indicating a unit of whole blood fromwhich the sheet was derived; (b) cross compatibility information foreach of the units of whole blood; and (c) a refrigeration unit for coldstorage of the plurality of plates.

Optionally, the cross compatibility information is provided as machinereadable data associated with the unique identifier in a look up tablein a memory of a computer.

Optionally, the cross compatibility information is provided as analiquot of un-clotted blood reserved from each of the units of wholeblood from which each of the plates in the plurality was prepared, eachaliquot associated cataloged by the unique identifier.

Optionally, at least some of the sterile plates of clotted whole bloodcomprise an external support structure in contact with the plate.

Optionally, at least some of the sterile plates of clotted whole bloodcomprise a mesh embedded in the plate.

According to some exemplary embodiments of the invention there isprovided a method of producing a biological dressing, comprising: a)spreading a unit of whole blood to cover an area while maintainingsterility; and b) allowing the blood to form a clot, typically in theform of a sheet.

Optionally, the method comprises adding a coagulation initiator prior tothe spreading.

Optionally, the coagulation initiator is selected to neutralize anactivity of an anticoagulant in the unit.

Optionally, the spreading occurs in a receptacle overlaid with a layerwhich prevents contact between the whole blood and the receptacle.

Optionally, the spreading is on a mesh which becomes embedded in theclot.

Optionally, the matrix adheres to the clot and contributes to atransferability thereof.

Optionally, the matrix is constructed of at least one material selectedfrom the group consisting of nylon, a polyurethane, a woven fabric, anon-woven fabric, silicon, rubber and organosilicone.

Some exemplary embodiments of the invention relate to use ofunfractionated whole blood in the manufacture and/or preparation of awound dressing.

According to some exemplary embodiments of the invention there isprovided method of treatment comprising: (a) providing an absorbentmatrix adapted for covering an area of skin of a subject in need oftreatment; (b) permitting an amount of whole blood to clot on saidmatrix to form a sheet of clotted whole blood thereon; and (c) applyingthe clotted blood-containing dressing on a skin injury.

Optionally, the blood includes autologous blood from the subject.

Optionally, the matrix includes a mesh.

Optionally, the mesh includes gauze.

Optionally, the method includes covering the sheet with a gas permeablematerial.

Optionally, the area of skin includes a non-bleeding wound (e.g. burnedarea or ulcer).

Optionally, the method includes covering the sheet with a conventionaldressing.

Optionally, the method includes maintaining sterility of the bloodduring the application.

According to some exemplary embodiments of the invention there isprovided a method for preparing plates of clotted whole blood, themethod comprising: (a) providing a block of clotted whole blood in acutting apparatus; (b) advancing the block incrementally; (c) cutting aslice from the block; and (d) alternately repeating (b) and (c).

In some exemplary embodiments of the invention, there is provided amethod of preparing a wound dressing including: (a) collecting a volumeof blood; and (h) clotting the blood ex vivo to form a clot.

Optionally, the method includes cutting a portion of the clot todimensions suitable for dressing a specific wound.

Optionally, the method includes providing a support structure upon whichsaid clot forms.

In some exemplary embodiments of the invention, there is provided a kitfor preparation of a wound dressing including: (a) a sterile containeradapted to receive a volume of blood; (b) sufficient anticoagulant toprevent the volume of blood from clotting; (c) sufficientanti-anticoagulant to cause the volume of blood to clot in the presenceof the anticoagulant; and (d) a tray adapted for spreading said volumeof blood to a desired thickness.

Optionally, the tray contains a support structure upon which said clotforms.

In some exemplary embodiments of the invention, there is provided a kitor assembly for preparing a dressing to be applied onto a skin injury,comprising one or more hardware components for processing a volume ofliquid whole blood to form a sheet of clotted blood.

Optionally, the sheet is of a defined thickness.

Optionally, the kit or assembly comprises one or more of: one or moreblood withdrawal devices; one or more blood collection receptacles; oneor more receptacles for receiving the blood and forming a clot thereon;one or more support matrices for the formed blood clot; ananticoagulant, optionally already included in the in the bloodwithdrawal or blood collection devices; and an anticoagulant inhibitorfor inducing clot formation.

In some exemplary embodiments of the invention, there is provided a kitfor preparation of a wound dressing, the kit comprising: (a) a sterilecontainer adapted to receive a volume of blood; (h) anticoagulant; (c)anti-anticoagulant; (d) a tray for receiving and spreading said volumeof blood to a desired thickness and for clotting whole blood therein;and (e) forming a wound dressing comprising the clot.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials are described below, methods and materials similar orequivalent to those described herein can be used in the practice of thepresent invention. In case of conflict, the patent specification,including definitions, will control. All materials, methods, andexamples are illustrative only and are not intended to be limiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying inclusion of the statedfeatures, integers, actions or components without precluding theaddition of one or more additional features, integers, actions,components or groups thereof. This term is broader than, and includesthe terms “consisting of” and “consisting essentially of” as defined bythe Manual of Patent Examination Procedure of the United States Patentand Trademark Office.

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of architecture and/or computer sci ence.

Percentages (%) of chemicals typically supplied as powders or crystals(e.g. EDTA and calcium salts) are W/V (weight per volume) unlessotherwise indicated. Percentages (%) of chemicals typically supplied asliquids (e.g. Citrate solutions and/or diluents) are V/V (volume pervolume) unless otherwise indicated.

BRIEF DESCRIPTION OF TILE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying figures.In the figures, identical and similar structures, elements or partsthereof that appear in more than one figure are generally labeled withthe same or similar references in the figures in which they appear.Dimensions of components and features shown in the figures are chosenprimarily for convenience and clarity of presentation and are notnecessarily to scale. The attached figures are:

FIG. 1A is a schematic diagram of a patient treated according anexemplary embodiment of the invention:

FIG. 1B is simplified flow diagram illustrating an exemplary treatmentmethod according to some exemplary embodiments of the invention;

FIG. 1C is simplified flow diagram illustrating adaptation of exemplarytreatment methods to existing treatment modalities;

FIG. 2 is simplified flow diagram illustrating another exemplarytreatment method according to some exemplary embodiments of theinvention;

FIG. 3 is a schematic diagram illustrating an article of manufactureaccording to some exemplary embodiments of the invention;

FIG. 4 is a schematic representation of a clot bank according to someexemplary embodiments of the invention;

FIG. 5 is a simplified flow diagram illustrating an exemplarymanufacturing method according to some exemplary embodiments of theinvention;

FIGS. 6A and 6B are schematic diagrams illustrating an apparatus forclot formation according to some exemplary embodiments of the inventionin two different operational states;

FIG. 7 is a schematic diagram illustrating another apparatus for clotformation according to some exemplary embodiments of the invention;

FIG. 8 is a schematic diagram illustrating another apparatus for clotformation according to some exemplary embodiments of the invention;

FIG. 9 is simplified flow diagram illustrating an exemplary method ofpreparing plates of clotted whole blood according to some exemplaryembodiments of the invention;

FIG. 10 is a photograph of an exemplary sheet of clotted whole blood ina receptacle according to an exemplary embodiment of the invention;

FIG. 11 is a photograph of an exemplary sheet of clotted whole bloodwith embedded support structure supported from the support structureaccording to an exemplary embodiment of the invention;

FIG. 12 is a photograph of exemplary sheets of clotted whole blood withembedded support structure depicting drainage of liquid from the plates;

FIG. 13 is a photograph of an exemplary sheet of clotted whole bloodaccording to an exemplary embodiment of the invention;

FIG. 14 is a photograph illustrating the sheet of FIG. 13 applied to askin surface according to an exemplary embodiment of the invention;

FIG. 15 is a photograph illustrating exudation of liquids from the sheetof FIG. 13 after application to the skin surface in FIG. 14; and

FIGS. 16A, 16B and 16C are a series of photographs illustratingadherence of the fully dried sheet to skin (FIG. 16A) and its removal bypeeling (FIGS. 16B and 16C) according to an exemplary embodiment of theinvention.

FIGS. 17A, 17B, 18A, 18B, 19A, 19B, 20A, 20B, 21A, 21B, 22A, 22B, 23Aand 23B are a series of photographs illustrating wounds prior totreatment (panel A in each figure) and after treatment with a wounddressing according to an exemplary embodiment of the invention (panel Bin each figure).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Embodiments of the invention relate to clotted whole blood containinganticoagulants and anti-anticoagulants, preparation of clotted wholeblood and/or use of clotted whole blood in medical treatment and/orpreparation of wound dressings and/or storage of clotted whole blood foruse as a wound dressing and/or apparatus for preparation of clottedwhole blood and/or application of whole blood to skin for in situ clotformation.

Specifically, some embodiments of the invention can be used to burnsand/or other skin injuries (e.g. chronic ulcers).

The principles of preparation and use of biological wound dressingsand/or apparatus to produce them and/or banks to store them and/ormethods of medical treatment relying upon them according to variousexemplary embodiments of the invention may be better understood withreference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Overview:

FIG. 1A depicts an exemplary embodiment 100 of the invention in which aportion of a skin surface 110 of a subject is covered with plates 120 ofclotted whole blood prepared specifically for that purpose. According tovarious exemplary embodiments of the invention plates 120 may beprovided in conjunction with a support structure 130 and/or covered witha conventional dressing 140. The portion of skin surface 110 of thesubject covered by plates 120 is selected because it requires treatment(e.g. a burned skin surface).

Plates 120 arranged on skin surface 110 form a biological dressing,whether prepare in advance, or allowed to form in situ at the treatmentarea. The various embodiments described hereinbelow are united by theidea that non-bleeding wounds (e.g. ulcers and/or burns) are deprived ofthe natural healing benefits of a scab formed by clotting blood.

Exemplary Treatment Method:

FIG. 1B is a simplified flow diagram of a method of treatment depictedgenerally as 102. Depicted exemplary method 102 includes preparing 122one or more plates of clotted whole blood and applying 132 applying theplate(s) to an area of skin of a subject in need thereof (e.g. a burnedor ulcerated area).

In some exemplary embodiments of the invention a support layer isadhered 142 to the sheet(s). Optionally, adhering 142 can occur duringor after preparation 122. In some exemplary embodiments of theinvention, the support layer is constructed to be gas permeable and/orliquid impermeable.

Alternatively or additionally, method 102 includes embedding 152 a meshwithin the plate. Typically, embedding is accomplished by contactingliquid whole blood with the mesh and allowing the blood to clot on themesh. Meshes suitable for this purpose include, but are not limited tofabric meshes (e.g. gauze or cheesecloth or non-woven fabrics).Optionally the mesh contains cotton fibers and/or cellulose.

Alternatively or additionally, method 102 includes covering 162 thesheet with a conventional dressing. The conventional dressing can serveto hold the plate in place and/or preserve sheet integrity and/or absorbexudates emanating from the sheet and/or prevent unwanted contact force(e.g. from donning and/or removing clothing). Optionally, theconventional dressing is changed with a same frequency or greaterfrequency than the sheet itself.

In some exemplary embodiments of the invention, sterility of the sheetis maintained from formation thereof until application to the skinsurface. Sterility can be maintained by any means known in the art.

According to various exemplary embodiments of the invention the sheetsare prepared by spreading whole blood in a layer with thickness of 1 mm,optionally 2 mm, optionally 3 mm or intermediate or greater or lesserthicknesses. A sheet of clotted blood will typically be less thick thanthe original layer of liquid blood.

Exemplary Use Scenarios

FIG. 1C is simplified flow diagram 104 illustrating adaptation ofexemplary treatment methods 102 to existing wet treatment 170 and drytreatment 180 modalities.

In some exemplary embodiments of the invention, a wet treatment mode 170is applied and the sheet is kept moist 124 after preparation 124. Themoist sheet is then applied 132 to an area of skin in need of treatment.Optionally, the sheet is covered 134 with a moist dressing and/orcovered 136 with a moisture retaining barrier.

In some exemplary embodiments of the invention, a dry treatment mode 180is applied and the sheet is dried 126 after preparation 124. The drysheet is then applied 132 to an area of skin in need of treatment.Optionally, the sheet is covered 133 with an absorbent dressing whichcan optionally be changed 135 so that fluid continues to be drawn fromthe sheet.

Optionally, a mixture of wet treatment 170 and dry treatment 180modalities is employed. In some exemplary embodiments of the invention,the sheet is kept moist 124, applied 132 and covered 133 with absorbentdressing. In some exemplary embodiments of the invention, the sheet isdried 126, applied 132 and covered with moist dressing 116.

Additional Exemplary Treatment Method:

FIG. 2 is a simplified flow diagram of another method of treatmentdepicted generally as 200. Depicted exemplary method 200 includesapplying 210 an absorbent covering to an area of skin of a subject inneed of treatment (e.g. a burned area). Optionally, the edges of theabsorbent covering are masked. Optionally, the absorbent coveringincludes a mesh such as, for example, gauze or cheesecloth. Opt the meshcontains cotton fibers and/or cellulose.

Depicted method 200 includes applying 220 whole blood to the absorbentcovering and allowing 230 the whole blood to clot to form a sheet ofclotted whole blood. If optional; masking has been employed, it canserve to direct applied blood preferentially towards the appliedabsorbent covering.

In some exemplary embodiments of the invention, method 200 includescovering the sheet with a gas permeable material (e.g. nylon and/orcotton and/or polyurethane).

In some exemplary embodiments of the invention, method 200 includescovering the sheet with a conventional dressing as described above formethod 102 and/or maintaining sterility of the blood during application220.

Exemplary Article of Manufacture:

FIG. 3 is a schematic representation of an article of manufacturedepicted generally as 300 viewed in cross section. Depicted exemplaryarticle of manufacture includes a sterile sheet 120 of clotted wholeblood. Sheet 120 includes an anticoagulant material dispersed throughoutthe sheet and a sufficient amount of anticoagulant inhibitor to permitformation of the sheet despite presence of the anticoagulant.Anticoagulants and inhibitors suitable for use in various embodiments ofthe invention are described hereinbelow.

Sheet 120 is provided in packaging material 350. Depicted exemplarypackaging material 350 includes a tearable seal 355 which allows removalof sheet 120 without tools. Packaging material 350 can be provide as anenvelope, a shrink wrap, a bubble pack, a sealed tub a jar, a canisteror any other packaging form known in the art.

According to various exemplary embodiments of the invention, article ofmanufacture 300 can be provided at ambient temperature, or providedunder refrigeration. Suitable refrigeration temperatures include 12, 8,4, 0, −20, −70 and −170 degrees centigrade and intermediatetemperatures.

According to various exemplary embodiments of the invention, sheet 120can include an internal support (e.g. mesh 330) and/or an externalsupport 340. Optionally, these support(s) can be used to facilitateremoval of sheet 120 from packaging material 350 and/or transfer ofsheet 120 to a desired treatment location and/or placement of sheet 120at the desired location.

Alternatively or additionally, internal support (e.g. mesh 330) and/orexternal support 340 can contribute to structural properties of sheet120. Structural properties include tensile strength, compressivestrength and elasticity.

Exemplary Storage Facility:

FIG. 4 is a schematic representation of a storage facility depictedgenerally as 400. Depicted exemplary storage facility 400 serves as abank of biological dressings. Facility 400 includes a plurality ofsterile sheets 121 of clotted whole blood, each sheet cataloged by aunique identifier (depicted as a bar code 450) indicating a unit ofwhole blood from which the sheet was derived. In the depicted exemplaryembodiment pates 120 are stored in groups of 5 within storage containers410. In the depicted embodiment, 5 sheets prepared from a single unit ofwhole blood are stored together and optionally treated as a singleinventory item or 5 separate inventory items. A single storage container410 is depicted in the inset in detail. In other embodiments of theinvention, sheets 120 are stored and catalogued individually.

Storage facility 400 includes a refrigerated compartment 440 containingsheets 20. In the depicted embodiment, 8 containers 410 are depicted incompartment 410 so that a total of 40 sheets are stored. This simplifiedrepresentation is solely for the sake of clarity of presentation. Inactual practice, the number of sheets 120 stored would probably be inthe thousands, tens of thousands or even hundreds of thousands.

Optionally, storage temperature and number of sheets stored areinterrelated. For example, lower storage temperatures may contribute tolonger “shelf life” of sheets 120 which, in turn, contributes to anability to accumulate a greater number of sheets before they must bediscarded.

Optionally, storage of a greater number of sheets contributes to anincreased likelihood of finding a suitable match for a subject in needof treatment.

Depicted exemplary storage facility 400 includes cross compatibilityinformation for each of the units of whole blood from which sheets 120are formed.

In some exemplary embodiments of the invention, the cross compatibilityinformation is provided as machine readable data associated with saidunique identifier (e.g. bar code 450) in a look up table in a memory 471of a computer 170. Optionally, a user enters cross compatibilityinformation from a subject in need of treatment via a suitable userinterface (e.g. keyboard 472) and is presented with a list of locations(e.g. on display 480) in refrigerated compartment 440 where suitablesheets 120 can be found. Suitable sheets in this case are “theoreticallysuitable” based upon a panel of markers. Optionally, a bar code reader(not shown) is used to verify identity of sheets 120 in specificcontainers 410.

Alternatively or additionally, cross compatibility information isprovided as an aliquot 430 of un-clotted blood reserved from each ofsaid units of whole blood from which each of sheets 120 was prepared.Aliquots 430 can be associated with unique identifier 450. In thedepicted embodiment, association is physical association resulting fromstorage in a same container 410 as corresponding sheets 120. Accordingto these embodiments, cross compatibility is checked practically bymixing un-clotted blood from a potential recipient from a sample drawnfrom aliquot 430 as is known in the art.

Optionally, theoretically suitable sheets are checked practically priorto actual use on a subject.

Optionally, at least some sheets 120 are provided on an external supportstructure in contact with the sheet and/or at least some of sheets 120include an internal support stricture (e.g. a mesh embedded in sheet120).

Exemplary Method of Manufacture:

FIG. 5 is a simplified flow diagram of a method of producing abiological dressing depicted generally as 500. Depicted exemplary method500 includes spreading 510 a standard unit of whole blood to cover anarea of at least 0.02 (e.g. 0.1 or more) square meters while maintaining520 sterility and allowing 530 the blood to form a clot. According tovarious exemplary embodiments of the invention, the area may be 0.15,0.2 square meters or greater or intermediate areas.

In some exemplary embodiments of the invention, method 500 includesadding a coagulation initiator to the whole blood prior to spreading510. According to various exemplary embodiments of the invention thecoagulation initiator is selected to neutralize an activity of ananticoagulant in the unit of whole blood.

Optionally, spreading 510 occurs in a receptacle overlaid with a supportlayer which prevents) contact between the whole blood and the receptacleas will be described in greater detail hereinbelow. Optionally, thesupport layer is gas permeable and/or liquid impermeable.

In some exemplary embodiments of the invention, spreading 510 isperformed on a mesh which becomes embedded in the clot.

In summary, exemplary embodiments of the invention encompass any use ofunfractionated whole blood in manufacture of a wound dressing.

Exemplary Apparatus for Clot Production:

FIGS. 6A and 6B are schematic diagrams illustrating a device forclotting whole blood ex-vivo according to some exemplary embodiments ofthe invention in two different operational states depicted generally as600 and 601.

In FIG. 6A, the device is depicted in fully assembled operational sttare600 in which it is connected to an external blood source 610 by aconnector 612. The depicted exemplary device includes an inlet 614comprising a channel of fluid communication to an interior of the deviceand a sample receptacle 650 adapted to spread a sample provided thereinto produce a uniform layer 620. Adaptation can include, for example,selection of a suitable material for construction to maintain bloodsurface tension at a value that encourages spreading. Alternatively oradditionally, a surface contacting the blood may be treated with asurfactant and/or textured to encourage spreading. In some exemplaryembodiments of the invention, receptacle 650 contains a sufficientamount of an anti-anti-coagulant to cause coagulation of bloodintroduced into the receptacle.

The depicted exemplary embodiment includes at least one supportstructure 640 deployed in receptacle 650 so that uniform layer 620 formson structure 640. Support structure 640 is depicted as an external layerwhich adheres to a blood clot formed thereupon. Optionally, the layer ispermeable to gas and/or impermeable to liquids.

Optionally, a space 642 is provided between receptacle 650 and layer 640for accumulation of liquids.

Alternatively or additionally, support structure 640 includes a meshembedded in layer 620 and clot sheet 120 resulting therefrom.

In the depicted embodiment, a removable cover 630 adapted to maintainsterility of layer 620 covers receptacle 650.

In some exemplary embodiments of the invention, the interior of thedevice is under vacuum pressure. According to various exemplaryembodiments of the invention interior volume of the device and/orrelative desired fill volume and/or flexibility of materials areconsidered when calculation an amount of vacuum to apply. Optionally,applied vacuum collapse, or partially collapses the device.

Exemplary Apparatus for Concurrent Production of Multiple Clots:

FIG. 7 is a schematic diagram illustrating a device depicted generallyas 700 for clotting whole blood ex-vivo to concurrently form multiplesheets. Depicted exemplary device 700 includes an inlet 710 comprising achannel of fluid communication to an interior of the device and aplurality of sample receptacles 750 (five are depicted, although othernumbers of receptacles may be employed) interconnected to allowconcurrent inflow from a common source. Each of receptacles 750 isadapted to spread a sample provided therein to produce a uniform layeras described hereinabove.

Depicted exemplary device 700 includes a sterility guard 730 adapted tomaintain sterility of the layer in each of stacked receptacles 750. Inthe depicted embodiment, receptacles 750 are configured as drawers sothat removal of one receptacle 750 does not effect sterility of saidlayer in any other of receptacles 750. Thus, at least some ofreceptacles 750 serve as sterility guards for another of receptacles750.

Alternatively or additionally, sterility guard 730 comprises a coverassociated at least some of receptacles 750.

In some exemplary embodiments of the invention, the interior of housing720 is provided under vacuum.

FIG. 8 is a schematic diagram illustrating a device depicted generallyas 800 for clotting whole blood ex-vivo to concurrently form multiplesheets. Depicted exemplary device 800 includes an inlet 810 providingfluid communication to an interior of housing 820.

The interior of housing 82(divided into a plurality of samplereceptacles interconnected to allow concurrent inflow from a commonsource by spacers 840. In the depicted embodiment interconnection is inparallel, although an alternate arrangement of spacers can be used toprovide receptacles in series. Series/parallel combinations are alsopossible in additional embodiments of the invention.

Each of receptacle is adapted to spread a sample provided therein toproduce a uniform layer as described above.

Cover 820 of housing 821 serves as a sterility guard adapted to maintainsterility of the layer in each of the receptacles.

During use, whole blood is loaded via inlet 810 and proceeds throughlumen 830 where it is in fluid communication with spaces between spacers840.

In the depicted exemplary embodiment, spacers 840 serve as externalsupport structure deployed in the receptacles. A layer of liquid wholeblood forms on each of support structures 840 which serve as externallayers to which a blood clot adheres as it forms thereupon.

In the depicted exemplary embodiment, mesh layers 330 are providedbetween spaces 840. Mesh layers 330 function as internal supportstructure embedded in the layer of liquid whole blood, and the bloodclot resulting therefrom.

In the depicted exemplary embodiment, device 800 includes an outletvalve 811 providing fluid communication between the interior of housing820 and an ambient environment. Optionally, valve 811 permits gas toexit the device as it fill with blood. In some exemplary embodiments ofthe invention, valve 811 is unidirectional and/or filter so that it doesnot compromise sterility.

In some exemplary embodiments of the invention, the interior of housing820 is provided under vacuum.

In the devices depicted in FIGS. 7 and 8, each the receptacles canoptionally contain a sufficient amount of an anti-anti-coagulant tocause coagulation of blood introduced therein.

Exemplary Method to Produce Sheets of Clotted Blood from a Block:

FIG. 9 is simplified flow diagram illustrating an exemplary method 900of preparing sheets of clotted whole blood according to some exemplaryembodiments of the invention. Depicted exemplary method 900 includesproviding a block of clotted whole blood in a cutting apparatus andadvancing 920 the block incrementally. After the incrementaladvancement, the block is cut 930 to form a slice which becomes a“sheet” as described hereinabove. Advancing 920 and cutting 930 can beincrementally repeated 940 to produce additional sheets.

Sheet thickness can be controlled by adjusting the increment ofadvancement 920.

Optionally, the block to be cut is frozen to increase hardness.

In some exemplary embodiments of the invention, a relatively smallvolume of blood (e.g. 10, 20, 30, 40 or 50 ml or intermediate volumes iscollected and clotted in an available container (e.g. test tube orsyringe barrel). Optionally, pieces, in the form of slices or sheets arecut from this clot using any available tools (e.g. a scalpel or razorblade. In some exemplary embodiments of the invention, cutting isperformed on a marked surface (e.g. graph paper) to keep slicesrelatively uniform in thickness. This type of method can be employed totreat ulcers with small sizes (e.g. 1, 2, 3, 4 or 5 square centimeter).

Exemplary Materials:

In some exemplary embodiments of the invention, external support layers(e.g. 340 and/or 640) can be constructed of one or more materialsincluding but not limited to nylon, polyurethane, cotton, a silicon andrubber. Optionally, materials are selected to be gas permeable and/orwater impermeable.

In some exemplary embodiments of the invention, sheet formingreceptacles (e.g. 650 and/or 750) can be constructed of one or morematerials including but not limited to glass, polyurethane,polyvinylchloride, polycarbonate, cellulose, polystyrene and a rubber.

In general, materials for construction of clotting devices describedhereinabove will be selected to be low cost, light weight andsterilizable. These factors all contribute to feasibility of productionof “single use” or “disposable” medical products.

In some exemplary embodiments of the invention, internal supports (e.g.330) are provided as meshes. Meshes suitable for this purpose include,but are not limited to fabric meshes (e.g. cotton gauze or cheesecloth).In other exemplary embodiments of the invention, non-woven fabrics aresubstituted for meshes. In other exemplary embodiments of the invention,the internal supports are provided as other types of matrices orscaffolds. Optionally, the mesh includes cotton fibers and/or cellulose.

Exemplary Dimensions:

In some exemplary embodiments of the invention, receptacles are sized tobe about 15×20 cm. If a layer of liquid whole blood 3 mm deep is placedin receptacles of this size, a standard unit of whole blood fills 5receptacles. The aggregate area of the 5 receptacles is 0.15 squaremeters, which corresponds to 10% of the skin surface of an averageindividual. A set of five receptacles arranged as deicted in FIG. 7 canbe prepared with a total height of about 5 to 8 cm and a footprint ofabout 16×21 cm or less.

If larger coverage areas are desired, thinner layers of whole bloodand/or a greater number of receptacles can be employed. Optionally,coverage of more than 0.15 square meters involves use of more than 1unit of whole blood.

In other exemplary embodiments of the invention, such as ulcers, smallercoverage areas are sufficient. Optionally, collection of small volumesof blood (e.g. 3, 5, 10, 20 or 30 cc or smaller or intermediate volumes)is sufficient to prepare a clot to cover an ulcer. In some exemplaryembodiments of the invention, correspondingly smaller clotting traysand/or thinner clots are employed.

Exemplary Anticoagulants and Anticoagulant Inhibitors:

Anticoagulants suitable for use in the context of the present inventioninclude, but are not limited to EDTA (ethylenediaminetetraacetic Acid),EGTA (ethylene glycol tetraacetic acid), Citrate (sodium citrate or acidcitrate dextrose), Heparin and oxalate.

Anticoagulants inhibitors suitable for use in the context of the presentinvention include, but are not limited to Ca²⁺, Mg²⁺, Kaolin, negativelycharged phospholipid (PL) and protamine sulfate. Table 1 summarizesexemplary ways in which clotting time can be controlled to obtain adesired working window (e.g. for preparation of sheets 120).

TABLE 1 Exemplary in vitro clotting times Tested material Clotting timeWhole blood 4-8 min Whole blood + EDTA or citrate Infinite Citratedwhole blood + Ca++ 2-4 min Citrated whole blood + PL (negatively charged60-85 sec phospholipids) + Ca++ Citrated whole blood + kaolin + PL +Ca++ 21-32 sec (aPTT) Citrated whole blood + thromboplastin + Ca++ 11-12sec (PT)

In some exemplary embodiments of the invention, whole blood with ananticoagulant added is obtained from a blood bank and sufficientanticoagulant inhibitor is added to achieve a clotting time of 2 to 8,optionally 4 to 8, minutes. This time is deemed sufficient to allowdistribution of the unit of blood in receptacles as a liquid forsubsequent clotting therein as described above.

Additional Exemplary Method

In some exemplary embodiments of the invention, a method of preparing awound dressing includes collecting a volume of blood and clotting theblood ex vivo to form a clot. Optionally, the collected volume is small(e.g. 5, 10, 15 or 20 ml or intermediate or lesser volumes). In someexemplary embodiments of the invention, the volume to be collected isdetermined in consideration of an area of a wound to be treated.Optionally, the method includes cutting a portion of the clot todimensions and/or shape suitable for dressing a specific wound. Cuttingmay be for example with scissors. Optionally, the method includesproviding a support structure upon which said clot forms. Supportstructures can be internal and/or external as described hereinabove.

Exemplary Kits:

Some exemplary embodiments of the invention relate to kits forpreparation of a wound dressing including clotted whole blood.Optionally, the kit includes a sterile, or sterilizable, containeradapted to receive a volume of blood and sufficient anticoagulant toprevent the volume of blood from clotting. The container can be, forexample, a syringe, a bag or test tube a vacuum filled test tube).

In some exemplary embodiments of the invention, the anticoagulant isprovided in the container. Optionally, the kit includes sufficientanti-anticoagulant to cause the volume of blood to clot in the presenceof the anticoagulant. Addition of the anti-anticoagulant to the bloodwith anticoagulant begins a window of time during which clotting occurs.During this window of time, the blood is transferred to a tray adaptedfor spreading the volume of blood to a desired thickness. Suitablethicknesses include 1, 2, 3, 4 and to 5 mm and intermediate or greaterthicknesses.

Optionally, the tray contains a support structure upon which said clotforms. Support structures can be internal and/or external as describedhereinabove.

Kits of this type can be used by individuals and/or by medicalpractitioners. Optionally, a user prepares several dressings at a timeand stores some or all of them for future use.

Exemplary Advantages:

According to various exemplary embodiments of the invention use ofclotted whole blood sheets 120 as described hereinabove promotes woundhealing and/or reduces incidence of infection and/or contributes to areduction in debridement frequency and/or reduces scarring. Theseadvantages are believed to be most significantly realized innon-bleeding wounds such as burns and chronic ulcers (e.g. diabetic footulcers). These advantages are believed to be available, at least to somedegree, without regard to the source of blood for sheets 120.

Exemplary Blood Sources

According to various exemplary embodiments of the invention blood usedto form sheets 120 can be autologous blood, homologous Whole bloodreceived from a blood bank or pooled homologous whole blood or pooledheterologous whole blood. It is generally accepted in medical practicethat a healthy individual can donate 1 unit of whole blood about every 6to 8 weeks without adverse effects. Alternatively, a single individualcan donate smaller amounts of blood at higher frequency.

In some exemplary embodiments of the invention, a subject in need oftreatment (e.g. a burn victim with burns over 8% of their body surface)donates a unit of whole blood which is subsequently used to form sheets120 using methods and/or devices as described hereinabove. Thistreatment strategy can contribute to a reduction in risks associatedwith incompatibility between blood donor and treated subject. However,there is a practical limitation as to how much skin surface can betreated with an autologous donation and/or whether a subsequentautologous donation will be available for continuation of therapy. Thesefactors will each, in turn, be influenced by thickness of sheets 120 asdescribed in greater detail hereinbelow.

In some exemplary embodiments of the invention, a subject in need oftreatment (e.g. a burn victim with burns over 16% of their body surface)is treated with sheets 120 prepared from homologous (e.g. human) bloodusing methods and/or devices as described hereinabove. This treatmentstrategy may slightly increase risks associated with incompatibilitybetween blood donor and treated subject. However, it is less limitedwith respect to how much skin surface can be treated and/or availabilityof additional sheets for continuation of therapy.

In other exemplary embodiments of the invention, homologous (e.g. human)blood from many units is pooled to form a large volume which issubsequently used to form sheets 120 to treat subjects in need thereofas described hereinabove. This treatment strategy is very amenable toindustrial scaling and reduces time and labor associated with crosscompatibility screening. However, a degree of risk associated withincompatibility between blood donor(s) and treated subject remains to beassessed. This treatment strategy is virtually unlimited with respect tohow much skin surface can be treated and/or availability of additionalsheets for continuation of therapy. There is a possibility that poolingof a large number of different types of blood prior to clotting to formsheets 120 may result in a “universal donor” situation as antibodies andantigens bind and neutralize one another prior to clot formation.

In other exemplary embodiments of the invention, heterologous (e.g.porcine or equine) blood from many units is pooled to form a largevolume which is subsequently used to form sheets 120 to treat subjectsin need thereof as described hereinabove. Alternatively or additionally,a pool of human blood may be extended by mixing with heterologous blood.This treatment strategy is also very amenable to industrial scaling andreduces time and labor associated with cross compatibility screening.However, a degree of risk associated with incompatibility between clotsderived from one or more heterologous species and treated subjectsremains to be assessed. This treatment strategy is also virtuallyunlimited with respect to how much skin surface can be treated and/oravailability of additional sheets for continuation of therapy. As withpooled homologous blood, there is a possibility that pooling of bloodfrom multiple unrelated species (e.g. porcine, ovine and equine andcanine blood) prior to clotting to form sheets 120 may result in a“universal donor” situation as antibodies and antigens bind andneutralize one another prior to clot formation.

Explanation of Sheet Thickness as a Treatment Limitation

In some exemplary embodiments of the invention, a unit of whole blood isspread over an area of 0.1 square meters or more and allowed to clot isdescribed hereinabove. The area which can be covered depends on desiredclot thickness after clotting. Table 2 summarizes exemplary coverageareas (% total body area) from a single standard unit of whole blood interms of initial layer thickness and resultant clot thickness.

TABLE 2 % body surface coverage as a function of liquid whole bloodlayer thickness Liquid blood thickness Clot thickness example (mm) (mm)Total area (M²) Body surface % 1 3 1.2 0.15 10 2 7 0.8 0.23 15 3 1.5 0.60.30 20 4 1 0.4 0.45 30 5 0.5 0.2 0.90 60

The summary in table 2 makes it clear that there is a linearrelationship between how much body surface can be covered by a unit ofwhole blood and the initial thickness of the whole blood layer used toprepare sheets 12. In some exemplary embodiments of the invention,internal support layers (e.g. mesh 330) and/or external support layers(e.g. 130 and/or 640) and/or conventional dressing (e.g. 140) contributeto a reduction in liquid blood thickness and/or clot thickness which arecompatible with therapy efficacy.

In the context of burn treatment, achievement of larger coverage areascan be critical. In the context of ulcer treatment, large coverage areais less important, although the relationship between coverage area andthickness remains.

General

It is expected that during the life of this patent many newanticoagulants and their corresponding inhibitors as well as polymericplastics will be developed and the scope of the invention is intended toinclude all such new technologies a priori.

As used herein the term “about” refers to ±10%.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

Specifically, a variety of numerical indicators have been utilized. Itshould be understood that these numerical indicators could vary evenfurther based upon a variety of engineering principles, materials,intended use and designs incorporated into the invention. Additionally,components and/or actions ascribed to exemplary embodiments of theinvention and depicted as a single unit may be divided into subunits.Conversely, components and/or actions ascribed to exemplary embodimentsof the invention and depicted as sub-units/individual actions may becombined into a single unit/action with the described/depicted function.

Alternatively, or additionally, features used to describe a method canbe used to characterize a wound dressing, an apparatus or kit or articleof manufacture and features used to describe a wound dressing, anapparatus or kit or article of manufacture can be used to characterize amethod.

It should be further understood that the individual features describedhereinabove can be combined in all possible combinations andsub-combinations to produce additional embodiments of the invention. Theexamples given above are provided solely for purposes of illustrationand are not intended to limit the scope of the invention which isdefined solely by the following claims. Specifically, the invention hasbeen described in the context of chronic ulcers and/or burn treatmentbut might also be used to treat other types of skin wounds.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

The terms “include”, and “have” and their conjugates as used herein mean“including but not necessarily limited to”.

Additional objects, advantages, and novel features of the presentinvention become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, various embodiments and aspects of the presentinvention as delineated hereinabove and as claimed in the claims sectionbelow find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Materials and Methods

The following materials and methods are used in performance ofexperiments described in examples hereinbelow:

Clotting trays were constructed of from disposable plastic food servicesheets with dimensions of size 1 cm×12 cm×8 cm for purposes ofexperimentation. In actual practice, other sizes and/or materials may beemployed. In some exemplary embodiments of the invention, Biocompatibleand/or sterilizable plastic polymers are employed.

Saran rap (polyvinylidene chloride [PVDC] made by S.C. Johnson & Son;USA) was used to cover clotting trays to reduce a tendency of clots tostick to the trays.

Surgical gauze (24×20 mesh, manufactured for M. Feingresh & Co. Ltd) wasemployed as an exemplary embedded support structure. A double layer ofcotton gauze was cut to 9 cm×13 cm or larger for the described clottingtrays.

Calcium Gluconate (B. Braun Melsungen A G; Germany) refers to 10 mlampoules of 10% containing Calcium Gluconate H2O 940 mg; CalciumD-Saccharate 4 H2O 50 mg and water for injection. Each 1 ml provides0.23 mol Ca++.

Kaolin (Imerys Minerals; Australia PTY) was used as a powder.

Blood was collected from a human volunteer using commercially availableblood donation bags with citrate anti-coagulant included. Fornon-citrated controls, blood was collected in a disposable syringe.Indicated volumes were placed in cups and mixed with anticoagulantinhibitors as indicated hereinbelow by swirling then immediately pouredinto clotting trays.

Blood donation bags were Sterile Non pyrogenic Single use infusion sets(MacoPharma; France) containing 63 ml CPDA1 (Formula for 1 L: Acidcitric Monohydr. 3.27gr; Natr. citras dihydr. 26.3 g; Mononatr. Phosphasdihydr. 2.51 g; Dextr. Monohydr. 31.9 g; Adenin HCL 0.349 g and Water to1000 ml [Ph 5.6+−0.3; 285 mmol Na/L]).

Example 1 Impact of Coagulants and Anticoagulants on Clot Formation

In order to assay the effects of citrate based anticoagulants andantagonists thereof on clot formation, various combinations were tested.Un-citrated whole blood (tray 2) served as a negative control. Citratedwhole blood with no antagonist (tray 1) served as a positive control.Experimental combinations of trays 3, 4, 5 and 6 are detailed in table3. Antagonist ingredients were mixed briefly in a plastic cup byswirling and poured over pre-cut surgical gauze placed in clotting trayslined with saran wrap to a depth of 4 mm. FIG. 10 is a photograph 1000of an exemplary clot in a clotting tray with embedded support structureprotruding from the edges 7 minutes after addition of calcium gluconate.At this time point, there was no visually perceptible difference betweentrays 3, 4, 5 and 6.

TABLE 3 Experimental coagulant/anticoagulant combinations Calcium BloodGluconate Tray volume (ml) citrated (ml) Kaolin 1 44 YES None None 2 44NO None None 3 40 YES 4 None 4 40 YES 8 None 5 40 YES 4 20 mg 6 40 YES 840 mg

Following clot formation, each clot was transferred by means of itsembedded support structure to a larger angled tray. FIG. 11 is aphotograph 1100 of one of the clots being transferred. Clots wereobserved for color and texture. Exuded fluid was characterized withrespect to volume and appearance. Negative control Tray 1 remainedun-clotted and was not involved in further manipulation and/orobservation.

FIG. 12 is a photograph 1200 of representative clots in angle trays.Fluid exudate is clearly visible in several trays.

After 12 Minutes from the time the calcium gluconate (and Kaolin whereapplicable) were added to the citrated blood:

Tray 2 contained a clot that was dark red, soft and thinner than theoriginal 4 mm liquid depth. About 10 ml-13 ml of red fluid exudatescollected in the angled tray and clotted.

Tray 3 contained a clot which was less dark red and more solid andthicker than the original 4 mm liquid depth. About 0.5 ml of red fluidexuded and did not clot.

Tray 4 was similar to tray 3.

Tray 5 was similar to trays 3 and 4, but the clot was more solid andthicker than trays 3 and 4. There was less volume of red fluid thantrays 3 and 4.

Tray 6 contained a clot that was darker, more rigid and thicker thanobserved in other trays. There was less red fluid exuded than all othertrays, and the color of the fluid was lighter than observed in othertrays.

After 23 Minutes from the time the calcium gluconate (and Kaolin whereapplicable) were added to the citrated blood, each clot was transferredby means of its embedded support structure to a clean larger angledtray. All clots where easily removable from their trays and kept theirshape during transfer.

Tray 2 contained a dark red clot which was not rigid and was about 2 mmor less in thickness. This clot continued to exude red fluid at a rateof about 1 ml/5 minutes. This clot exhibited axial translation withrespect to the embedded support structure indicating an insufficientdegree of adhesion.

Trays 3, 4, 5 and 6 the clots were a lighter red than tray 2 and werevery solid and thicker than the original 4 mm liquid depth. Smallamounts of pale red fluid continued to exude.

Tray 6 contained a clot with the best performance in terms of shaperetention, thickness preservation, rigidity and fluid retention.

After 37 minutes each clot was transferred by means of its embeddedsupport structure to a scale for weighing. The citrated clots of trays3, 4, 5 and 6 weighed about 30% moiré than the positive control clot oftray 2.

Based upon these observations, 8 cc calcium gluconate does not seem toimprove clot properties relative to over 4 cc calcium gluconate

Kaolin, at either amount, seems to improve clot properties.

In this experiment, properties of the fresh whole blood clot (Tray 2)where judged inferior to citrated coagulated whole blood clots.

About 42 Minutes after addition of anticoagulant antagonists:

FIG. 13 is a photograph 1300 of the clot of Tray 6. This clot was placedin a standard freezer (−20 degrees C.) for 5 days, taken out and placedon the skin of the thigh and fixed in place with medical adhesive, andcovered with layers of cotton.

FIG. 14 is a photograph 1400 of the clot of Tray 3 which wassubsequently placed on the skin of the thigh and fixed in place bylayers of gauze held by adhesive tape and covered with layers of cottonheld with additional adhesive tape.

All other clots where left to dry in open air

Preliminary testing of clotted whole blood as a wound dressing materialis described below in Examples 2 and 3.

Results presented in this example indicate that embedded supportstructure were useful in preserving clot integrity and/or shape and/orin facilitating transfer and/or handling.

This example illustrates that it is feasible to prepare sheets ofclotted whole blood in desired sizes and shapes for use in variousexemplary embodiments of the invention as described hereinabove.

Example 2 Exemplary Use of a Sheet of Freshly Clotted Blood inPreparation of a Wound Dressing

In order to evaluate the possibility of using sheets of clotted wholeblood as part of a wound dressing, the clot from tray 3 was placed onthe skin (FIG. 14) of the thigh and covered with layers of surgicalgauze and then layers of cotton held in place with adhesive tape.

After 12 hours, the cotton was removed. FIG. 15 is a photograph 1500showing that exudates from the clot has soaked through the surgicalgauze laid over the clot. It appears that clot exudates soaked into theoverlaid gauze made this material part of the clot layer.

This example illustrates that clotted whole blood adheres well to intactskin. The intact skin is used as a model for non-bleeding wounds (e.g.burns and/or ulcers) so the results confirm feasibility of using clottedwhole blood as a dressing for non-bleeding wounds. Optionally, dressingsaccording to exemplary embodiments of the invention can be applied tobleeding wounds as well

Example 3 Effect of Blood Clot Applied to Intact Skin Surface

Following example 2, the clot was manually removed from the intact skinsurface to which it had been applied. FIG. 16A is a photograph 1600showing the dried clot adhered to the skin. FIG. 16B is a photograph1602 peeling of the dried clot from the skin. FIG. 16C is a photograph1604 showing that removal of the dried clot from to the skin caused noperceptible irritation. Removal was manual by peeling and gentle pullingas seen in FIG. 16C.

The clot was thin and completely dry at this stage. Small defects in theform of holes were observed in the clot. The holes appeared tocorrespond to places where the internal support structure (gauze) hadbeen broken or torn.

This example illustrates that use of sheets of clotted whole blood inthe context of burn treatment provides an alternative to conventionalmethods. Optionally, sheets of clotted whole blood contribute to areduction in wound debridement.

Example 4 Exemplary Use of a Sheet of Frozen Clotted Blood inPreparation of a Wound Dressing

In order to demonstrate that clots can be prepared in advance and storedfor later use as wound dressings, the clot from tray 6 (FIG. 13) wasstored frozen for 5 days then thawed at room temperature. The clot wasused to prepare a wound dressing as in Example 3. Results weresubstantially the same as in example 3.

This example illustrates that storage of blood clots prior to their useas wound dressings according to exemplary embodiments of the inventionis feasible.

Example 5 Additional Observations on Air Dried Blood Clots

in order to evaluate the potential of storing clotted whole blood underambient conditions, clots from trays 2, 4 and 5 were left to dry in openair for 25 hours. Compared to the bandaged clots, the air dried clotswere thicker, although they became completely dry. These clots werecharacterized by a very dark red color and were without defects (holes).These clots were odor free.

Example 5 illustrates that it is feasible to store clotted whole bloodat ambient temperatures for at least 1 day. The absence of odor suggestsno putrefaction. It is not clear whether the absence of holes resultsfrom a decrease in handling or other factors.

In general the air dried clots were thicker and maintained more bloodelements than those applied as bandages (examples 3 and 4).

Example 6 Treatment of Actual Wounds in Human Subjects

In order to confirm that clotted whole blood can be efficaciouslyemployed as a wound dressing, human subjects with actual wounds ofvarious types were recruited and treated using dressings preparedsubstantially as described above.

Consecutive patients referred to the Palliative care department and tothe nursing departments of the Shoham Geriatric Medical Center(Pardes-Hanna; Israel) were screened for recruitment between June 2009and December 2009. All potential subjects were already under treatmentfor their wounds with previously accepted standard of care treatment.The study was approved by Institution review board. In total 7 woundswith etiology stages 2-3 from 6 patients (2 males and 4 females) weredressed clotted whole blood dressings according to exemplary embodimentsof the invention. Subjects or their legal guardians signed informedconsent forms prior to inclusion in the study. Recruitment criteria wereage 18-100, wounds stages 2-3 and informed consent. Exclusion criteriawere wounds larger than 8 cm by 8 cm. Cancerous wounds, inability toprovide 30 cc blood per week and a proven sepsis established by a bloodculture in the past 2 weeks. All ongoing wound localized care treatmentswere stopped prior to initiating treatment according to an exemplaryembodiment of the invention. Wound characteristics are summarized intable 4. The Typing and staging was according to previously publishedcriteria (NPUAP pressure ulcer staging 2007, [npuap.org];. Preventionand treatment of skin tears K. Leblanc et al, Wound Care Canada, Volume6, Number 1, 2008).

TABLE 4 Wound characteristics Type/ Wound Age/Gender etiology LocationSize cm² Stage 1 93/F Tear wound Left lower leg 28.1 3 2 88/F Venousulcer Right foot back 3.12 NA and fistula 3 88/F Venous ulcer Left upperfoot 1.6 NA 4 48/F Pressure Right heel 3.61 3 ulcer 5 32/M AmputatedMiddle finger 0.89 3 finger tip 6 90/M Pressure Left heel 4.7 3 ulcer 765/F Traumatic Right Shin 2.8 2 wound

Dressings based upon clotted whole blood were prepared using a kitaccording to an exemplary embodiment of the invention. The exemplary kitcontains 3 sizes of sterile single use clotting trays, small (14.5 cm²),medium (26.4 cm²) and large (64 cm²). Each clotting tray is fitted withmedical cotton gauze stretched across the bottom of the tray. Asexplained hereinabove the gauze becomes embedded in the clot as itforms. Optionally, the gauze contributes to robustness of the dressingand/or simplifies transfer of the dressing to the wound and/or makestrimming to shape easier.

For each wound treatment, wound size was determined by measuring themaximum length between two points on the wound edges. According to thedetermined size, an appropriate tray was selected (e.g. by the nurse).The clotting tray was selected to be larger than the wound in order toensure that the clotted whole blood dressing would cover the whole woundarea and beyond, at least 0.5 cm of the intact skin around the wound.Specific instructions for the amount of CPDA1, whole blood and thecoagulation activating calcium and kaolin mixture were provided as partof the kit for each of the three sizes of clotting trays: small, mediumand large. The exemplary amounts are CPDA1 (1.5 ml, 2 ml and 4 ml),amount of whole blood to be extracted (10 ml, 13 ml and 30 ml) andamount of coagulation activator, the calcium and kaolin mixture (2 ml,2.5 ml and 5 ml).

Optionally, preparation of the wound bed for dressing was conductedaccording to accepted procedures, e.g. washing with sterile salineand/or debridement depending on wound type and/or stage. The patient andwound current and past clinical status were documented and the wound wasphotographed while holding a single use ruler next to the wound edges asa means of monitoring treatment progress.

A syringe (20 ml or 50 ml syringe according to the selected tray) wasfilled with the appropriate amount of CPDA1 and used to extract venousblood from the patient. Blood withdrawal and handling was performedaccording to the Hospital precautions guidelines.

In parallel, 5 ml calcium gluconate (B. Braun, Germany) was mixed in asterile vial with 35 mg of sterile kaolin white powder (Merck). Thedesired amount (as described above) of coagulation activating mixturewas extracted from the vial using the syringe containingblood/anticoagulant mixture. The contents of the syringe were gentlymixed for 10 seconds to induce coagulation.

At this point, the coagulating blood was transferred into the preselected clotting tray and the tray was placed on a flat horizontalsurface. The nurse then waited (e.g. about 10 minutes) for the dressingincluding a sheet of clotted whole blood to form. At this stage, theclotting tray is opened using sterile gloves and clotted whole bloodbased dressing is transferred to the wound, placing it on the wound sothe embedded pad faced upwards. The dressing according to an exemplaryembodiment of the invention was then affixed to the wound by covering itwith a non adhesive pad, then gauze and then it was wrapped by anelastic bandage or by using a Band Aid™ (Johnson & Johnson, Somerville,N.J.).

Following initial treatment, a follow up program including two types ofvisits was implemented. In a first “monitoring” type of visit, theexemplary whole blood clot based dressing was not removed from thewound. In a second “re-application” visit the exemplary whole blood clotbased dressing was removed and a new whole blood clot based dressing wascreated and applied as described above.

Monitoring visits were conducted every 2 days. In these sessions thedressing according to an exemplary embodiment of the invention was notremoved, but only the gauze pads above the clot. The visit includedvisual inspection of the exposed surface of the dressing according to anexemplary embodiment of the invention to document the adherence of theclot to the wound, visual inspection of the wound periphery for adverseevents and olfactory inspection to monitor for possible infection.

Regular re-application visits were scheduled every 6 to 8 days dependingon the staff availability. The dressing changes were pre-scheduled inorder to document the wound healing process underneath. In the twotrauma wounds treated, the clotted whole blood dressing was left on thewound until it naturally dropped off. These wounds were not chroniculcers. Re-application visits included documentation, photographing (ofthe existing dressing and the wound being treated) and removal andreplacement of the clotted whole blood based dressing according to anexemplary embodiment of the invention. The Re-application visit alsoincluded wound bed preparation according to standard procedures usinggauze and saline.

In this study, treatment continued until complete healing of the woundor until clinical determination that the wound could not further improvewithout additional invasive procedures, such as surgery of a fistula,procedures which are not performed in the geriatric center.

All kit components and materials were supplied as sterile single useitems with regulated and pre-measured materials. The whole kit isdisposable and was discharged according to the hospital regulations.Throughout the study measures were taken to prevent the clot fromgetting wet (e.g. using plastic wrap such as CLING-FILM™ or SARAN WRAP™to wrap the limbs and specially dedicated waterproof adhesive pads toprotect the wound during bathing).

Wounds were photographed during the healing process using a digitalcamera (Cannon Power Shot A590) using a flash, at a defined distance of50 cm from the wound, facing the front of the wound. In each photographa disposable reference ruler was placed in close proximity to the woundedges. The ruler included the date, patient number and wound studynumber as filled in by the research staff.

The wound pictures where analyzed by using NIH Image analysis software“ImageJ” (National Institute of Health, Bethesda, Md., USA) which isavailable for download at http://rsb.info.nih.gov/ij/download.html. Thissoftware is in the public domain. According to other exemplaryembodiments of the invention, commercial image analysis softwareproducts are employed instead of image J.

Image analysis in this case included:

a. measurement of the number of pixels in 10 mm as presented in thepicture reference ruler.

b. delineations of the wound edges and documentation of automatic pixelsarea output of the image.

c. repeating step b three times.

d. calculating the average pixels area of the 3 separate delineations,

e. transformation of the pixels area to mm² area by the measured pixelsper 10 mm,

f. documentation of the final result in the patients CRF for thespecific date the picture was taken.

Optionally, other image analysis strategies can be substituted for thisstrategy.

The clinical results of dressing wounds with clotted whole blooddressings according to exemplary embodiments of the invention arepresented in table 5. Five out of seven wounds were totally healed andthe remaining two were mare than 80% healed. Five out of seven woundswere moderately to heavily exudating at the onset of treatment withdressings according to exemplary embodiments of the invention. In all ofthe exudating wounds, the extraction of the exudates through the clottedwhole blood based dressing and its absorbance in to the covering padswas documented. This demonstrates that dressings according to exemplaryembodiments of the invention are not occlusive. On average there was adressing application every 8.8 days. The cases are presented below.

FIG. 18 illustrates a case of a vascular wound diagnosed with anexudating fistula 1 cm next to the main wound which required surgicalprocedure before treatment (FIG. 18A) and after treatment (FIG. 18B).The photographs indicate that the main wound showed complete and goodhealing, while the exudating fistula did not completely heal although itwas covered with the same dressing according to an exemplary embodimentof the invention for the same period of time. Therefore the total woundincluding the fistula was calculated as only 80% healed.

FIG. 19 illustrates a case of a venous ulcer on the left upper footprior to treatment (FIG. 19A) which was completely healed (FIG. 19B) by11 days of treatment with a dressing according to an exemplaryembodiment of the invention.

FIG. 20A depicts a pressure ulcer in which treatment was stopped after82% closure (FIG. 20B), due to re-opening of the wound that resultedfrom recurrent direct pressure on the wound when it was not dressed withthe dressing according to an exemplary embodiment of the invention.

The two trauma wounds, a case of left shin laceration (tear wound) in a93 year old woman (FIG. 17A pre-treatment) and a right hand middlefinger tip amputation in a 34 years old male (FIG. 21A pre-treatment)had a single application of the dressing according to an exemplaryembodiment of the invention, which lasted for 21 days (laceration) and19 days (amputation), until the dressing dropped off, healing wasachieved without the need of further dressing (see FIGS. 17B and 21Brespectively).

There were two cases of ulcers in patients with diabetes, one pressureulcer on the left heel (FIG. 22A pre-treatment) and the other atraumatic wound on the right shin (FIG. 23A), the healing of ulcers indiabetic patients was similar to the healing on non diabetic patients(See FIGS. 22B and 23B respectively),

TABLE 5 Treatment Results Size (cm²) Size (cm²) Days Wound pre-treatmentpost-treatment (applications)* FIG. 1 28.1 0 21 (1) 17 2 3.12 0.71 61(7) 18 3 1.09 0 11 (1) 19 4 3.61 0.64 36 (5) 20 5 0.89 0 19 (1) 21 6 4.70 49 (7) 22 7 2.8 0  7 (1) 23 *days dressed with clotted whole blooddressing (number of dressing applications)

Results presented in this example clearly demonstrate that a careprovider can effectively and safely prepare a clotted whole blooddressing according to exemplary embodiments of the invention, optionallyfrom a kit provided at the point-of-care and containing pre-measuredcomponents and/or tools associated with preparation of the dressing.

Alternatively or additionally, results presented in this example clearlydemonstrate that dressing wounds with a dressing including an autologousblood clot is safe for use in chronic wounds of various etiologies. Nodelay in wound healing was observed and no adverse events (e.g. sepsisand/or inflammation) were observed during the study. Results summarizedin table 5 suggest that dressings according to exemplary embodiments ofthe invention actually accelerate wound healing relative to previouslyavailable standard of care dressings.

Alternatively or additionally, results presented in this example clearlydemonstrate that that for the 7 wounds treated with a clotted wholeblood dressing according to exemplary embodiments of the invention, thedressing promoted healing.

What is claimed is:
 1. A method of wound dressing of a wound of asubject, the method comprising: a. spreading a volume of whole bloodonto a support matrix; b. allowing the volume of whole blood to clot onthe support matrix to form a wound dressing comprising clotted wholeblood; and c. applying the wound dressing comprising the clotted wholeblood onto the wound.
 2. The method of claim 1, wherein spreading of thevolume of whole blood onto the support matrix is at a locationphysically separated from the wound.
 3. The method of claim 1, whereinspreading of the volume of whole blood onto the support matrix is to adesired thickness.
 4. The method of claim 1, wherein spreading of thevolume of whole blood is in an amount that allows the support matrix tobe embedded in the whole blood clot.
 5. The method of claim 1, whereinprior to spreading the volume of whole blood onto the support matrix,the volume of whole blood is mixed with a coagulation initiator.
 6. Themethod of claim 5, wherein the coagulation initiator comprises kaolin.7. The method of claim 6, wherein an amount of kaolin is sufficient toimprove clot properties on the support matrix.
 8. The method of claim 1,wherein the volume of whole blood is obtained from blood withdrawn fromthe subject in need of the wound dressing.
 9. The method of claim 8,wherein the volume of whole blood withdrawn from the subject in need ofthe wound dressing is mixed with an anticoagulant prior to spreading thevolume of whole blood onto the support matrix.
 10. The method of claim9, wherein the volume of whole blood withdrawn from the subject in needof the wound dressing is mixed with a coagulation initiator prior tospreading onto the support matrix.
 11. The method of claim 10, whereinthe coagulation initiator is kaolin.
 12. The method of claim 1, whereinthe volume of whole blood is a whole blood unit.
 13. The method of claim1, wherein the wound is a chronic wound.
 14. The method of claim 13,wherein the chronic wound is a non-bleeding wound.
 15. The method ofclaim 14, wherein the non-bleeding wound is selected from one of a skinburn and a chronic ulcer.
 16. A method of improving treatment of a woundcomprising: applying onto a wound of a subject in need of treatment awound dressing comprising a support matrix carrying clotted whole blood,the treatment being improved as compared to a wound dressing without theclotted whole blood.
 17. The method of claim 16, wherein the wound is achronic wound.
 18. The method of claim 17, wherein the chronic wound isa non-bleeding wound.
 19. The method of claim 14, wherein thenon-bleeding wound is selected from one of a skin burn or a chroniculcer.